Research Item (9)
- Feb 2016
Both phosphatidylserine (PS) and oligomannose/desialylated glycans are known to be exposed on the surface of dying cells and used by macrophages to endure effective efferocytosis. Recognition of phosphatidylserine by annexin A5 is widely implicated in various technologies aimed to isolate apoptotic cells. Our recent finding suggested that recognition of phosphatidylserine and apoptotic glycans by macrophages is of complementary nature. Here we developed lectin-conjugated magnetic microparticles recognizing specific glycans of apoptotic cells and its use for detection and separation of dying cells. We conjugated Narcissus poeticus lectin, specific to oligomannose N-glycans, and Viscum album agglutinin, specific to desialylated glycans to the surface of superparamagnetic and fluorescent-superparamagnetic microparticles, and tracked their binding to subpopulations of human polymorphonuclear leukocytes undergoing apoptosis. Glycan targeting microparticles were effectively found on apoptotic cells, while specific lectin inhibitors of carbohydrate nature (like heptylmannoside), displaced dying cells from complexes with microparticles, suggesting a reversible binding. Simultaneous application of microparticles conjugated with annexin A5 and Viscum album agglutinin exhibited additive effect on the efficiency of apoptotic cells removal from population.
Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G–Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin–Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum. Copyright © 2014 John Wiley & Sons, Ltd.
- Feb 2014
Recently, we reported the first known incidence of antibodies possessing catalytic sialidase activity (sialidase abzymes) in the serum of patients with multiple myeloma and systemic lupus erythematosus (SLE). These antibodies desialylate biomolecules, such as glycoproteins, gangliosides and red blood cells. Desialylation of dying cells was demonstrated to facilitate apoptotic cell clearance. Here we assessed the possibility of facilitating dying cell clearance with the use of F(ab)2 fragments of sialidase abzymes. Two sources of sialidase abzymes were used: 1) those isolated from the sera of patients with SLE after preliminary screening of a cohort of patients for sialidase activity; 2) by creating an induced sialidase abzyme through immunization of a rabbit with a synthetic hapten consisting of a non-hydrolysable analogue of the sialidase reaction conjugated with BSA or KLH. Antibodies were purified by ammonium sulfate precipitation, protein-G affinity chromatography and HPLC-SEC. The effect of desialylation on efferocytosis was studied using human polymorphonuclear leukocytes, both viable and aged, as prey, and human monocyte-derived macrophages. Treatment of apoptotic and viable prey with both disease-associated (purified from the blood serum of SLE patients) and immunization-induced (obtained by rabbit immunization) sialidase abzymes, their F(ab)2 fragments and bacterial neuraminidase (as positive control) significantly enhanced the clearance of prey by macrophages. We conclude that a sialidase abzyme can serve as a protective agent in autoimmune patients and that artificial abzymes may be of potential therapeutic value.
Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.
Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.
p>Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 184.108.40.206, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.</p
- Apr 2009
Constantly increasing sector of industry of biomedical diagnos-tics requires photosensitive and stable nanomaterials which can be specifically bio-labeled for targeting selected cells in the body. Here we describe two types of novel nanomaterials conjugated with spe-cific proteins recognized by dying (apoptotic and necrotic) cells. They are luminescent GaN:Eu 3+ -doped nanoparticles with wide green emission and very narrow red emission and fluorescent green-emitting fluorescein-containing polystyrene nanoparticles. These nanoparticles were conjugated with carbohydrate recogniz-ing proteins – the lectins – that are targeting specific cell surface glycoproteins which were recently shown to be novel markers of dying cells (Bilyy et al., 2003, Bilyy et al., 2007). Both luminescent and fluorescent nanoparticles possessed high brightness and low level of photobleaching and efficiently interacted with dying cells, thus discriminating them from the intact ones.